Recent genetic studies have revealed that malignant gliomas in children and young adults often show shared missense mutations, which encodes the replication-independent histone 3 variant H3.3. Approximately 30% of overall glioblastoma and 70% of diffuse intrinsic pontine glioma (DIPG) cases harbor the amino acid (AA) substitution from lysine (K) to methionine (M) at position 27 of H3.3 (K27M mutation, hereafter). The K27M mutation in DIPG is universally associated with shorter survival compared with patients with non-mutated H3.3. We recently found that a peptide that includes the K27M mutation (H3.3.K27M epitope, hereafter), can induce specific cytotoxic T lymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2.1+ donor-derived CD8+ T-cells. Induced CTLs recognize the H3.3.K27M epitope that is endogenously expressed by HLA-A2.1+ glioma cell lines that also harbor the K27M mutation. We will evaluate our hypothesis that the H3.3 K27M represents a novel, shared neoantigen epitope for effective and safe T-cell-based immunotherapy. We will evaluate the following three specific aims which are directed towards the development of safe and effective immunotherapy for patients with K27M mutated glioma. Aim 1. Determine the preclinical safety and potency of synthetic peptide encoding the H3.3.K27M epitope for development of vaccine strategies. We will determine preclinical efficacy and safety of vaccinations with the synthetic peptide using HLA-A2.1 transgenic mice. We will also determine whether elongation of the original H3.3.K27M epitope, specifically to include helper T-cell epitope, improves the efficacy of the synthetic peptide-based vaccination. Aim 2. Characterize CTL clones raised against the H3.3. K27M for key AA residues and possible cross-reactivity to naturally existing variants of H3.3. We have already obtained T-cell clones reactive to the H3.3.K27M epitope and will determine the key epitope residues within the H3.3.K27M epitope by alanine scanning. We will evaluate whether our CTL clones are cross-reactive to naturally existing variants of the H3.3.K27M epitope. Aim 3. Determine the specific anti-glioma activity of T-cells transduced with T-cell receptor (TCR) against the H3.3.K27M epitope. We have already cloned ?- and ?-chains of TCRs from our CTL clones. Using T-cells transduced with recombinant TCR vector, we will determine whether the transduced TCR is functional and mediate specific (without off-target effects) anti-glioma responses against the H3.3.K27M epitope. We will also evaluate whether TCR-transduced T-cells are properly activated to show favorable phenotype for therapy of brain tumors and can mediate preclinical efficacy in vivo. Impact. Outcomes from these studies will provide us with a strong basis for developing peptide-based vaccine (Aim 1) as well as adoptive cell therapy using autologous T-cells transduced with the TCR (Aims 2 and 3).